PROJECT 3: SUMMARY/ABSTRACT Nathan K. LeBrasseur, M.S., Ph.D. As a mechanistic complement to the human projects (Projects 1 and 2) in the Mayo Clinic Specialized Center of Research Excellence (SCORE) on Sex Differences, this Project will test the central hypothesis that ovariectomy (OVX)-induced endocrine disruption in female mice hastens and exacerbates the accumulation of age-related senescent cells and, in turn, compromises clinically-relevant measures of physical and cognitive performance. Our hypothesis is founded on our recent work demonstrating the causal role of cellular senescence, a hallmark of aging, in the genesis of multiple age-related conditions. Our preliminary data support our hypothesis and demonstrate that senescent cells mediate functional decline and, in specific tissues relevant to physical and cognitive performance, are more abundant in female compared to male mice of advanced age. We will test our central hypothesis through two specific aims, which will (1) determine the degree to which OVX and long-term estrogen replacement impacts healthspan in mice; and (2) determine the extent to which OVX and long-term estrogen replacement affects the accumulation and abundance of senescent cells in multiple tissues. Our approach will leverage a novel transgenic reporter system in mice that enables the unique ability to quantitatively, temporally, and inducibly visualize, track, and isolate p16Ink4a- positive senescent cells. At six months of age, mice will undergo OVX or sham surgery and begin continuous estrogen or placebo treatment [three experimental groups: 1) sham surgery + placebo, 2) OVX + estrogen, and 3) OVX + placebo]. We will then measure the trajectory of healthspan across five clinically-relevant domains: body composition, physical performance, cardiovascular function, metabolic homeostasis, and cognitive function at 12, 18, and 24 months of age. We will systematically quantify senescent cells at these timepoints across tissues, and test their association with measures of healthspan. We anticipate that OVX mice will exhibit accelerated and more severe deficits in the five healthspan domains compared to sham-operated mice, and that estrogen replacement will both delay and mitigate functional consequences of OVX. We predict that the deleterious effects of OVX and therapeutic effects of estrogen will be reflected, if not preceded, by senescent cell burden in tissues responsible for synchronizing functional parameters. As in Project 1 for women, we further anticipate that circulating markers of systemic senescent cell burden will correlate with the clinically-relevant measures of physical and cognitive health in mice. This Project will enable systematic exploration of the long-term effects of OVX and estrogen replacement on integrated measures of healthspan and importantly, will reveal whether aging and endocrine disruption exert synergistically detrimental effects on the fundamental biology of aging at a level of resolution not achievable in humans.